INTRODUCTION
Diabetes mellitus (DM) is the leading cause of chronic kidney disease (CKD) in the USA and worldwide. An estimated 422 million adults are living with diabetes globally, and up to 40% may develop CKD during their lifetime.[ 1 ] Diabetic kidney disease (DKD), a severe and common microvascular complication of DM, has become a major cause of end-stage renal disease (ESRD).[ 2 ] The progression of DM to DKD before a clear diagnosis of diabetes poses a unique treatment challenge. The general treatment options for DKD are mainly focused on glycemic and hypertensive control and pharmacotherapy, which includes angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter 2 (SGLT2) inhibitors.[ 3 ] However, combination therapy of ACEIs and ARBs are not recommended due to the increased risk of hyperkalemia and/or acute kidney injury (AKI). As the glucose-lowering effect of SGLT-2 inhibitors is dependent on renal function, they are not recommended in patients with an estimated glomerular filtration rate (eGFR) of less than 30 mL/min/1.73m2 .[ 4 ] Due to these treatment limitations, a need exists for complementary and alternative treatments to delay the progression of kidney damage in patients with DKD.
With a history of several thousand years and rapid development over the past decades, traditional Chinese medicine (TCM) has shown significant therapeutic effects in the treatment of DKD.[ 5 ] The Huangkui capsule (HKC) or Huangshukuihua capsule has recently been used as adjuvant therapy for patients with AKI or CKD arising from DKD, alleviating proteinuria and hematuria and improving renal function by inhibiting immunoreaction, inflammatory damage, and interstitial fibrosis.[ 6 , 7 ] HKC administration for the management of DKD appears clinically feasible due to its satisfactory clinical efficacy and safety profile. However, the lack of evidence-based studies supporting its use remains unclear. Furthermore, due to the complex components in HKC, the multiple targets and pathways involved in the treatment of DKD have not been elucidated. Recently, network pharmacology has been used to study the complex relationship between protein/gene and disease and predict the targets and pathways of medicine and diseases, which could completely reveal the underlying mechanism of drug-diseases interactions. Therefore, this study conducted a meta-analysis and utilized network pharmacology to analyze the efficacy of HKC in DKD to provide valuable information for making clinical decisions.
METHODS
Search strategy
All references with the theme that evaluated the efficacy and safety of HKC combined with ACEI/ARB on DKD were searched from the following databases: China National Knowledge Infrastructure (CNKI), Wanfang, Chongqing VIP, SinoMed, Web of Science, EMBASE, PubMed and The Cochrane Library. The time range of data retrieval was from the inception date till August 2022. Search terms included “Huangkui capsule”, “okra capsule”, “Abelmosk capsule”, “Diabetic Nephropathies”, “Diabetic Kidney Disease”, “angiotensin-converting enzyme inhibitor”, “angiotensin receptor blocker”, “ACEI”, “ARB”, “Randomized controlled trial”, “RCT” and other synonyms. A detailed retrieval strategy has been provided in supplementary materials, https://links.lww.com/IMNA/A3 Chinese Journal of Integrated Traditional Chinese and Western Medicine Nephropathy , the Journal of Traditional Chinese Medicine , and so on.
Inclusion criteria
Types of studies
All randomized controlled trials (RCTs) using HKC combined with ACEI/ARB to treat DKD patients were accepted, regardless of their languages.
Types of participants
Patients with diabetes who were diagnosed with DKD were enrolled without restrictions based on their race, region, gender, age, and disease course. The diagnostic criteria for DM were according to the guidelines for the prevention and treatment of DM established by the Diabetes Society of the Chinese Medical Association or World Health Organization. DKD was identified by either microproteinuria, massive proteinuria, or eGFR less than 60 mL/min/1.73m2 in diabetic patients after ruling out other causes of CKD.
Interventions
The combination of HKC and ACEI/ARB (HKC+ACEI/ARB) was the experimental group, and ACEI/ARB was designated the control group. Both groups received the same therapy, including medication dosage and other therapies, except HKC.
Outcomes
The primary outcome was urine albumin excretion rate (UAER), and the secondary outcomes were 24 h-urinary total protein (UTP), serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels. Adverse effects were also assessed.
Exclusion criteria
Patients
Patients who did not meet the inclusion criteria and those who had undergone kidney transplants or hemodialysis were excluded.
Types of studies
Non-randomized controlled clinical trials, such as animal experiments, retrospective studies, and case reports, were excluded. Additionally, low-quality articles were excluded.
Interventions
Studies involving treatments combined with other Chinese medicine therapies were excluded. Studies using different drug dosages or unclear medications in the HKC combined with ACEI/ARB and ACEI/ARB groups were excluded.
Outcomes
Studies with incomplete data or non-relevant outcomes were excluded.
Study selection and data extraction
Two researchers (Yunling Geng and Zhaocheng Dong) selected references independently according to the inclusion and exclusion criteria, compared differences, discussed, and finally enrolled all the studies used in this meta-analysis. Then, patients' baseline information, intervention methods, medicine dosage, and outcomes were extracted from the studies.
Assessment of bias
Based on the Cochrane handbook, we assessed “random sequence generation”, “allocation concealment”, “blinding of participants and personnel”, “bling of outcome assessment”, “incomplete outcome data”, “selective reporting”, and “other bias”.
Statistical analysis
RevMan 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen) was used for statistical analysis. Mean difference (MD) was used for continuous variables, and relative risk (RR) was used for dichotomous variables. We evaluated 95% confidence intervals (CI) and tested the heterogeneity results. When the result was P > 0.05 and I 2 < 50%, there was no statistical heterogeneity among each group, and the fixed-effects model was adopted. Otherwise, a random-effects model was used. P < 0.05 was considered statistically significant. We performed a subgroup analysis to rule out the cause of heterogeneity. Funnel plots of primary outcome evaluated the analysis of publication bias.
Target gene prediction of the ingredients of HKC
The main chemical components of HKC were analyzed and identified using high performance liquid chromatography (HPLC). Then the SwissADME database (http://www.swissadme.ch http://www.swisstargetprediction.ch https://www.uniprot.org
Prediction of DKD-related target genes
Four gene-disease databases were used to predict DKD-related target genes, including OMIM (https://www.omim.org https://go.drugbank.com https://www.genecards.org https://www.disgenet.org Homo sapiens setting. The UniProt database was used as above. After collecting all gene names, we removed the duplicate data. Finally, we obtained DKD-related target genes.
Building drug-ingredients-targets-disease network
Venny 2.1 website (https://bioinfogp.cnb.csic.es/tools/venny/
Construction of protein-protein interaction (PPI) network
We constructed a PPI network using the STRING database (https://cn.string-db.org
Enrichment analysis of GO and KEGG pathway
The gene function annotation and pathway analysis of target genes were performed in the DAVID database (https://david.ncifcrf.gov
RESULTS
References Search
A total of 381 references were retrieved from eight literature databases, and four were obtained from several core journals by manual searching. Exactly 153 articles were screened through titles or abstracts after removing duplicates. Following the inclusion and exclusion criteria, 127 articles were excluded, including 85 low-quality studies, 18 non-RCT articles, 12 studies involving combination with other drugs, six articles with no relevant outcomes, and six articles that did not study HKC and DKD. After reading the full text, another 11 articles were excluded owing to variable drug doses, unclear diagnostic criteria, meta-analysis, and unrelated outcomes. Finally, 15 studies were included in this meta-analysis review [Figure 1 ].[ 8–9 ]
Figure 1.: Study flow diagram. RCT, randomized controlled trial; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; CNKI, China National Knowledge Infrastructure.
Study characteristics
Exactly 15 studies with 1255 patients were included, with 637 patients in the experiment group and 618 in the control group.[ 8–9 ] Three articles enrolled DKD patients with hypertension, cardiovascular disease, hyperlipidemia, and other chronic diseases.[ 15 , 19 , 21 ] The characteristics of all patients are demonstrated in Table 1 , and all met the diagnostic criteria.
Table 1 -
Characteristics of all included patients
References
Participants
No. of participants (T/C)
Sex (M/F)
Age (y)
Disease course (y)
Intervention
Control
Duration
Outcomes
Chen et al . 2014 [22 ]
DKD
75/75
T: 38/37
T: 58.1 ± 10.9
T: 6.1 ± 3.9
C+HKC 2.15 g tid
Conventional therapy + ACEI 10 mg qd
8 weeks
SCr, BUN,24h-UTP
Dai et al . 2017[21 ]
DKD
40/40
T: 19/21
T: 46.21 ± 12.13
T: 5.4 ± 3.9
C+HKC 2.15 g tid
Conventional therapy + ARB 80 mg/d
3 months
UAER, BUN, SCr
Ding et al . 2019[20 ]
DKD
55/55
T: 21/24
T: 56.35 ± 6.34
T: 7.61 ± 1.69
C+HKC 2.5 g tid
Conventional therapy + ARB 80 mg/d
3 months
24h-UTP, SCr
Gao et al . 2017[19 ]
DKD
40/40
T: 26/14
T: 67.39 ± 4.72
T: 7.23 ± 1.29
C+HKC 2.5 g tid
Conventional therapy + ARB 80 mg qd
8 weeks
24h-UTP, BUN, SCr, UAER
Gao 2017[15 ]
DKD
40/40
T: 29/11
T: 52.64 ± 9.15
T: 9.16 ± 1.01
C+HKC 2.5 g tid
Conventional therapy + ARB 0.15 g qd
16 weeks
BUN, SCr, 24h-UTP, UAER
Hu et al . 2011[18 ]
DKD
40/40
-
-
-
C+HKC 2.15 g tid
Conventional therapy + ARB 80 mg qd
56 days
24h-UTP, BUN, SCr, TG, TC
Jia 2015[10 ]
DKD
38/32
T: 27/11
T: 50.30 ± 15.50
T: 6.0 ± 5.6
C+HKC 2 g tid
Conventional therapy + ARB 4 mg qd
4 weeks
24h-UTP, SCr,LDL
Li et al . 2009[16 ]
DKD
40/40
T: 26/14
42–65 (52.1)
6–15 (8.5)
C+HKC 2.15 g tid
Conventional therapy + ACEI 10 mg qd
8 weeks
24h-UTP, SCr, BUN, TC, TG
Sun et al . 2012[14 ]
DKD
45/45
T: 29/16
T: 62.34 ± 12.18
T: 10.47 ± 4.26
C+HKC 2.15 g tid
Conventional therapy + ACEI 10 mg qd
12 weeks
24h-UTP, SCr, BUN
Wang et al . 2010[9 ]
DKD
32/31
T: 16/16
T: 58.89 ± 15.98
8.5 ± 1.5
C+HKC 2.15 g tid
Conventional therapy + ARB 80 mg/d
16 weeks
BUN, SCr, 24h-UTP, UAER
Xiao et al . 2010[13 ]
DKD
33/32
T: 17/16
T: 57.98 ± 16.07
8.5 ± 1.5
C+HKC 2.15 g tid
Conventional therapy + ARB 80 mg/d
16 weeks
BUN, SCr, 24h-UTP, UAER
Xu et al . 2013[8 ]
DKD
36/25
32/29
36 ± 14.5
7.5 ± 4.5
C+HKC 2.15 g tid
Conventional therapy + ARB 150 mg qd
4 weeks
UAER, SCr
Xu et al . 2016[11 ]
DKD
62/62
T: 36/26
T: 43.8 ± 2.9
T: 1.48 ± 0.32
C+HKC 2.15 g tid
Conventional therapy + ARB 80 mg/d
6 months
TG, TC, LDL-C,BUN, SCr,24h UAER
Xu et al . 2018[12 ]
DKD
19/19
T: 10/9
T: 54.14 ± 10.26
T: 5.7 ± 4.1
C+HKC 2.15 g tid
Conventional therapy + ACEI 10 mg qd
56 days
SCr, BUN,24h-UTP
Zhu 2017[17 ]
DKD
42/42
T: 25/17
T: 59.37 ± 7.52
T: 8.71 ± 2.38
C+HKC 2.5 g tid
Conventional therapy + ARB 80 mg/d
8 weeks
BUN, SCr, UAER
DKD, diabetic kidney disease; T, treatment group; C, control group; HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; UAER, urinary albumin excretion rate; 24h-UTP, 24hour-urine total protein; BUN, blood urea nitrogen; SCr, serum creatinine; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol.
Assessment of bias
Risk of bias assessment
According to the Cochrane handbook, we assessed all 15 studies, and the assessment results are shown in Figure 2 . Only six studies using randomized digital tables were evaluated as low-risk.[ 11 , 12 , 14 , 19 , 21 , 22 ] The remaining nine studies that described non-standard or non-detailed randomized methods were assessed as unclear risk. None of those studies mentioned allocation concealment and the performance of blinding outcome assessment, so they were rated low risk. Owing to the special dosage form of HKC, it was hard to perform blinding of participants and personnel; thus, they were rated high risk. All studies had reported complete outcomes with no dropout, and the attrition bias was evaluated as low risk. For reporting bias, 10 studies that reported detailed outcomes and adverse effects were assessed as low risk, while the remaining five studies did not mention the numbers of outcomes in both experimental and control groups. For other biases, studies with more complete outcomes were rated low risk. Eight studies that showed detailed clinical experimental data were rated as low risk, the six studies that did not mention adverse effects were assessed as unclear risk, and one study with unstated baseline data was rated as high risk.[ 18 ]
Figure 2.: Bias risk assessment. (A) Risk of bias graph: review authors' judgments about each risk of bias item presented as percentages across all included studies. (B) Risk of bias summary: review authors' judgments about each risk of bias item for each included study. “+” means “low risk of bias”, “?” means “unclear risk of bias”, and “-” means “high risk of bias”.
Outcomes
UAER
Since one study used a different unit, standard mean difference was used to describe the difference in UAER between the HKC combined with ACEI/ARB group and the ACEI/ARB group. A random-effects model was used due to the heterogeneity test results (P < 0.00001, I 2 = 97%). Eight studies reported changes in UAER among the groups. Compared with the control group using ACEI/ARB only, ACEI/ARB combined with HKC significantly lowered UAER (SMD = −1.92, 95% CI [−2.93, −0.92], P = 0.0002) [Figure 3 ].
Figure 3.: Forest plot of HKC comparison: HKC+ACEI/ARB group vs. ACEI/ARB group. The forest plot shows the difference in UAER between the HKC+ACEI/ARB and ACEI/ARB groups. Heterogeneity test results are expressed as I 2 and P values. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; UAER, urine albumin excretion rate.
24h-UTP
Ten studies reported change in 24h-UTP levels, with six studies using ARB and four using ACEI. Following the heterogeneity test results (P < 0.00001, I 2 = 99%), a random-effect model was used. Compared with the ACEI/ARB group, the HKC+ACEI/ARB combined group could reduce 24h-UTP levels (MD = −0.41, 95% CI [−0.64, −0.18], P = 0.0004). Considering the effects of different medications on analysis, a subgroup analysis was used to assess the effects of HKC on the 24h-UTP level. The results showed that HKC combined with ARB or ACEI reduced 24h-UTP levels compared with using ARB or ACEI only (MD = −0.43, 95% CI [−0.73, −0.14], P = 0.004, and MD = −0.40, 95% CI [−0.49, −0.30], P < 0.00001, respectively) [Figure 4 ].
Figure 4.: Forest plot of ACEI/ARB subgroup: HKC+ARB group vs. ARB group, and HKC+ACEI group vs. ACEI group. The forest plot shows the difference in 24h-UTP levels between HKC+ACEI/ARB and ACEI/ARB groups. Heterogeneity test results are expressed as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; 24h-UTP, 24h urinary total protein.
SCr
All 15 studies reported change in SCr levels, consisting of 11 studies using HKC+ARB and four using HKC+ACEI. A random-effects model was used due to the heterogeneity test results (P < 0.00001, I 2 = 88%). Compared with the control group using ACEI/ARB only, the experimental group using ACEI/ARB combined with HKC reduced SCr levels (SMD = −0.68, 95% CI [−1.02, −0.34], P < 0.0001). Due to the effects of different medications on experimental outcomes, a subgroup analysis was used. Compared with the control group using ARB or ACEI only, the experimental group combining HKC to the ARB or ACEI groups led to a reduction in SCr levels (SMD = −0.71, 95% CI [−1.14, −0.28], P = 0.001 and SMD = −0.60, 95% CI [−1.19, −0.02], P = 0.04, respectively) [Figure 5 ].
Figure 5.: Forest plot of ACEI/ARB subgroup: HKC+ARB group vs. ARB group, and HKC+ACEI group vs. ACEI group. The forest plot shows the difference in SCr between the HKC+ACEI/ARB and ACEI/ARB groups. Heterogeneity test results are expressed as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; 24h-UTP, 24h urinary total protein, SCr, serum creatinine.
BUN
Twelve studies reported BUN levels, including eight studies using ARB and four using ACEI. A random-effects model was used following the heterogeneity test results (P < 0.00001, I 2 = 91%). Compared with the control group using ACEI/ARB only, ACEI/ARB combined with HKC reduced BUN levels (SMD = −0.59, 95% CI [−1.04, 0.14], P = 0.01). Subgroup analysis was used to reduce the effects of the confounding factor caused by different drugs. Interestingly, compared with the control group using ARB only, HKC+ARB did not reduce BUN levels (SMD = −0.62, 95% CI [−1.25, 0.02], P = 0.06). Changes in BUN levels in ACEI combined with HKC were not significantly different (SMD = −0.54, 95% CI [−1.17, 0.09], P = 0.10) [Figure 6 ].
Figure 6.: Forest plot of ACEI/ARB subgroup: HKC+ARB group vs. ARB group, and HKC+ACEI group vs. ACEI group. The forest plot shows the difference in BUN between the HKC+ACEI/ARB and ACEI/ARB groups. Heterogeneity test results were expressed as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; BUN, blood urea nitrogen.
TC
Three studies reported changes in serum TC levels. A random-effects model was used due to the heterogeneity test results (P < 0.00001, I 2 = 95%). Compared with the control group, using ACEI/ARB combined with HKC reduced TC levels (MD = −1.22, 95% CI [−2.04, 0.39], P = 0.004) [Figure 7 ].
Figure 7.: Forest plot of HKC comparison: HKC+ACEI/ARB group vs. ACEI/ARB group. The forest plot showed the difference in TC between the ACEI/ARB combined with HKC group and the ACEI/ARB group. Heterogeneity test results were expressed as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; 24h-UTP, 24h urinary total protein; TC, total cholesterol.
TG
A random-effects model was used according to the heterogeneity test results (P = 0.01, I 2 = 77%). Compared with ACEI/ARB only, TG levels were reduced in the HKC+ACEI/ARB group (MD = −0.54, 95% CI [−0.94, −0.15], P = 0.007) [Figure 8 ].
Figure 8.: Forest plot of HKC comparison: HKC + ACEI/ARB group vs. ACEI/ARB group. The forest plot shows the difference in TG between the HKC + ACEI/ARB and ACEI/ARB groups. Heterogeneity test results are described as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; TG, triglyceride.
LDL-C
Two studies reported LDL-C levels. A random-effects model was used due to the heterogeneity test results (P = 0.04, I 2 = 76%). Compared with the control group, no significant difference in LDL-C levels was observed in the HKC + ACEI/ARB group (MD = −0.45, 95% CI [−1.21, 0.31], P = 0.24) [Figure 9 ].
Figure 9.: Forest plot of HKC comparison: HKC + ACEI/ARB group vs. ACEI/ARB group. The forest plot shows the difference in LDL-C between the HKC + ACEI/ARB and ACEI/ARB groups. Heterogeneity test results are expressed as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol.
Adverse effects
Seven studies reported adverse effects, and none of the patients withdrew during the experimental period. The adverse effects included gastrointestinal reactions, nervous system response, and cough, which disappeared after receiving proper treatment. Following the heterogeneity test results (P = 0.69, I 2 = 0), a fixed-effect model was used. Compared with using ACEI/ARB only, the combination of HKC with ACEI/ARB showed no significant difference in adverse effects (RR = 0.98, 95% CI [0.43, 2.22], P = 0.96). The subgroup analysis revealed that compared with the control group using ARB or ACEI only, HKC combination with ARB or ACEI showed no statistical significance in adverse effects (RR = 2.23, 0.5; 95% CI [0.59, 8.48], [0.16, 1.61]; P = 0.24, 0.24, respectively) [Figure 10 ].
Figure 10.: Forest plot of ACEI/ARB subgroup: HKC + ARB group vs. ARB group, and HKC + ACEI group vs. ACEI group. The forest plot shows the difference in adverse effects between the HKC+ACEI/ARB and ACEI/ARB groups. Heterogeneity test results are expressed as I 2 and P value. HKC, Huangkui capsule; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.
Publication bias
Based on the funnel plots of UAER, there was a high risk of publication bias for the asymmetrical outcomes [Figure 11 ].
Figure 11.: Funnel plot of UAER for assessing publication bias in the included studies. UAER, urine albumin excretion rate. SMD, standard mean difference; SE, standard error of mean.
Prediction of target genes of HKC ingredients and DKD
We analyzed and identified 20 main components in HKC. After screening using the SwissADME database, only five components showed high gastrointestinal absorption, including gallic acid, 5-(hydroxymethyl)-2-furancarboxylic acid, protocatechuic acid- 3-O-β-D-glucoside, protocatechuic acid, and quercetin. Then we predicted the targets of five active ingredients with the SwissTargetPrediction database and transformed those target proteins into target genes using the UniProt database. After deleting the duplicate data, we obtained 262 target genes of HKC ingredients.
Meanwhile, we searched DKD-related targets from databases with the Homo sapiens setting (OMIM 256, DrugBank 41, GeneCards 1721, DisGeNet 1189). The UniProt database was used to transform data from DrugBank. Exactly 2459 target genes were obtained after removing duplicate data.
Finally, we used Venny 2.1 website to take the intersection. A total of 127 genes were obtained, which werea the common target genes of HKC and DKD. The results are shown in Figure 12A .
Figure 12.: The predictions of target genes and construction of drug-disease network and PPI network. (A) Venn diagram of HKC-DKD common target genes. The blue circle represents the target genes predicted for HKC ingredients (2459 target genes in list 1). The yellow one represents target genes predicted for DKD (262 target genes in list 2). The intersection represents the common target genes of HKC and DKD (127 target genes). (B) HKC-ingredients-targets-DKD network and protein-protein interaction network. HKC, Huangkui capsule; DKD, diabetic kidney disease; PPI, protein-protein interaction.
Establishment of HKC-ingredients-targets-DKD network
The active ingredients in HKC were abbreviated due to their lengthy names. Gallic acid was assigned the number one (HKC-1); 5-(hydroxymethyl)-2-furancarboxylic acid was assigned the number two (HKC-2); protocatechuic acid - 3-O-β-D-glucoside was assigned the number three (HKC-3); protocatechuic acid was assigned the number four (HKC-4), and quercetin was assigned the number five (HKC-5). After importing into Cytoscape 3.7.2, the HKC-ingredients-targets-DKD network was constructed, as shown in Figure 12B .
PPI network
The 127 common targets were imported into the STRING database with the Homo sapiens setting and highest confidence (0.900). The PPI network is shown in Figure 12B . After importing the data into Cytoscape software, we used CentiScaPe 2.2 to filter the target genes, setting the proximity, betweenness, and degree to at least or equal to the median value, to obtain eight key target genes. The key targets were SRC, AKT1, HSP90AA1, PIK3R1, SYK, FYN, ESR1 and F2.
GO analysis and KEGG pathway enrichment analysis
The common target genes were imported into the DAVID database to perform GO and KEGG pathway enrichment analysis. In the biological process, there were 348 items, including response to drug, positive regulation of protein kinase B signaling, response to xenobiotic stimulus, negative regulation of the apoptotic process, positive regulation of smooth muscle cell proliferation, etc . In the cellular component, there were 79 items, including the plasma membrane, extracellular exosome, membrane raft, receptor complex, etc . In molecular function, 132 items were obtained, including transmembrane receptor protein tyrosine kinase activity, identical protein binding, protein tyrosine kinase activity, enzyme binding, etc . The top 10 items of biological process, cellular components, and molecular function are shown in Figure 13A .
Figure 13.: Enrichment analysis of GO and KEGG pathway. (A) Top 10 items in biological process (shown in blue), cellular component (green), and molecular function (red). (B) Top 20 pathways from KEGG analysis. PI3K-Akt, HIF-1, AGE-RAGE, Rap1, and VEGF were involved in the treatment of DKD. KEGG, Kyoto Encyclopedia of Genes and Genomes; DKD, diabetic kidney disease; GO, Gene Ontology; PI3K-Akt, phosphatidylinositol-3-kinase-serine-threonine kinase; HIF-1, hypoxia inducible factor-1; AGE-RAGE, advanced glycation end product-receptor for advanced glycation end product; Rap1, ras-proximate-1; VEGF, vascular endothelial growth factor.
Exactly 98 pathways were identified in the KEGG pathway enrichment analysis, including the PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, endocrine resistance, lipid metabolism and atherosclerosis, cancer pathways, etc . The top 20 pathways are shown in Figure 13B .
DISCUSSION
DKD, a kidney disease of metabolic origin, has high morbidity and mortality in diabetic patients, which is the primary cause of ESRD globally. The characteristics of patients with DKD are microalbuminuria, heavy proteinuria, or reduction in eGFR after ruling out other causes of CKD.[ 23 ] For many years, the therapeutic agents inhibiting the renin-angiotensin-aldosterone system (RAAS), ACEI, and ARB, were the only agents available for treating DKD.[ 24 ] The Kidney Disease: Improving Global Outcomes (KDIGO) guidelines emphasize the management of blood pressure, with ACEI/ARB, recommended as the first choice. Many clinical trials have reported that ACEI/ARB could help to prevent the development and progression of DKD and avoid the incidence of other serious conditions, such as cardiovascular events.[ 25–26 ] ACEIs lower blood pressure by promoting sodium and water excretion by inhibiting aldosterone and improving the vasodilatation of renal blood vessels. ARBs bind to angiotensin receptors to accelerate the excretion of sodium and water. Although ACEI/ARB has shown an established role in reducing ESRD and preventing the progression of DKD, it does not lower cardiovascular or all-cause mortality.[ 28 ] Meanwhile, since ACEI/ARB cannot block the RAAS completely, a dual-blockade therapy using ACEI and ARB was proposed.[ 29 ] However, a study reported that despite dual blockade in reducing proteinuria, it could not achieve long-term benefits compared with a single drug. In addition, the increased adverse effects profile limited the use of dual therapy.[ 30 ] Thus, searching for new drug combinations is urgent.
HKC, extracted from Abelmoschus manihot (L. ) medic, has been used to treat DKD for many years.[ 31 ] However, the potential effects and mechanism of HKC combined with ACEI/ARB for halting DKD have not been demonstrated clearly. This meta-analysis aimed to provide high-quality evidence for the clinical application of HKC. As shown above, ACEI/ARB combined with HKC could remarkably lower UAER, and 24h-UTP, TC, and TG levels. Although the results showed statistical significance in SCr and BUN, the changes made no sense in the clinic. In terms of adverse effects, the combination group showed no significant difference compared with ACEI/ARB. In conclusion, compared with ACEI/ARB, the combination of HKC and ACEI/ARB could reduce proteinuria and the progression of DKD with a good safety profile. Moreover, the effects of HKC showed no significant difference between the combination of ACEI and ARB. Recently, increasing studies on HKC for the treatment of DKD have been reported. HKC plays a role in kidney protection, involving several pathways, including inflammation-related signals, oxidative stress-related pathways, and metabolic pathways.[ 31–32 ] In this study, network pharmacology revealed that the underlying mechanism of HKC treatment for DKD was mainly associated with the PI3K-Akt signaling pathway, and 21 genes were involved in this signaling pathway, including GSK3B, HSP90AA1, SYK, FLT4, INSR, PIK3R1, EGFR, IL2, PIK3CG, PTK2, IGF1R, RXRA, CDK2, KDR, BCL2, AKT1, TEK, MET, TLR4, MCL1, and BCL2L1. According to the PPI network, the key genes, including AKT1, HSP90AA1, PIK3R1, and SYK, were involved in the PI3K-Akt signaling pathway. Consistent with our findings, studies have shown that HKC could alleviate early glomerular pathological changes such as the thickening of glomerular basement membrane (GBM), glomerular hypertrophy, and mild mesangial expansion by inhibiting the activity of phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (Akt)/mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) signaling pathways.[ 34 ]
Additionally, accumulated evidence has demonstrated that HKC could ameliorate proteinuria induced by primary glomerular diseases, such as primary immunoglobulin A nephropathy and membranous nephropathy.[ 35 ] Proteinuria, a symptom of various diseases, is usually caused by metabolic and immune-mediated disturbances of glomerular filtration. Generally speaking, the pathogenesis of metabolic-mediated proteinuria is complicated, including but not limited to glomerular hyperfiltration, highly increased trans-glomerular pressure, and the consequent glomerular and tubulointerstitial damage.[ 36 ] Notably, the reduction in trans-glomerular pressure and hyperfiltration benefits the attenuation of proteinuria, which has been found in clinical trials with the administration of ACEI, ARB, SGLT2 inhibitors, and finerenone.[ 37–38 ] The underlying mechanism of glomerular hyperfiltration at the early stage of DKD might be the excess glucose reabsorbed by the proximal tubules via SGLT2 and the reduction in distal sodium delivery, which decreases tubulo-glomerular feedback with consequent dilation of afferent arterioles and constriction of angiotensin-induced efferent arterioles. In the EMPA-REG OUTCOME, CREDENCE, and other trials, SGLT2 inhibitors have shown beneficial renoprotective effects in proteinuric CKD.[ 41 ] An equally remarkable drug is finerenone, a selective mineralocorticoid receptor antagonist reported to reduce albuminuria in patients with DKD.[ 42 ] However, the clinical trials of HKC in combination with SGLT2 inhibitors and finerenone are not very satisfactory and require further investigation. Of note, immune-mediated proteinuria is characterized by the disruption of the glomerular filtration barrier caused by immune complex deposition within the glomerular basement membrane and consequent activation of the complement cascade, podocyte damage, and a series of inflammatory injuries.[ 43 ] Thus, agents of immune modulation, such as cyclosporine, and inflammation reduction, such as corticosteroids, could effectively prevent proteinuria.
This meta-analysis had several limitations. The heterogeneity test results showed significant differences making the conclusions less reliable. Several sources of heterogeneity limit our confidence in the conclusions. First, the low quality of clinical trials and small qualified sample size contributes to the wide disparity in the heterogeneity test results, indicating that more high-quality and large-sample-size RCTs are needed to provide high-quality evidence. Second, ACEI and ARB drugs are the general terms for two major categories of drugs, including benazepril, captopril, valsartan, and so on, which may influence the pooled analysis result. Third, the wide age range, disease course, different doses, and duration of medication may result in greater heterogeneity. Other limitations include: Since only published articles were included, there is a high risk of publication bias and other biases, such as performance bias, that all studies had not demonstrated the blinding method and distribution hiding. Notably, more limitations exist in the network pharmacology of HKC due to the absence of experimental validation. Although the key target genes and crucial pathways of HKC-DKD have been predicted, we cannot draw solid conclusions as we cannot guarantee the reliability of the results. Besides, since the components of HKC were analyzed and identified for only 20 genes, the number of prediction targets was insufficient.
CONCLUSION
The combination of ACEI/ARB and HKC could improve the prevention of DKD by lowering UAER, 24h-UTP, TC, and TG levels with a good safety profile compared to ACEI/ARB only. Furthermore, HKC may treat DKD primarily by inhibiting the PI3K-Akt signaling pathway, and further experimental studies are required.
Conflicts of interest
Ping Li is the Editor-in-Chief of the journal and Wei Jing Liu is an Editorial Board Member of the journal. The article was subject to the journal's standard procedures, with peer review handled independently of these members and their research groups.
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